Proliferative diabetic retinopathy is associated with a low level of the natural ocular anti-angiogenic agent pigment epithelium-derived factor (PEDF) in aqueous humor. a pilot study.

Retinopathy is the most common microvascular diabetes complication and represents a major threat to the eyesight. The aim of this study was to address the role of pro- and anti-angiogenic molecules in diabetic retinopathy in the aqueous humor of the eye. Aqueous humor was collected at cataract surgery from 19 diabetic patients and from 13 age- and sex-matched normoglycemic controls. Levels of pro-angiogenic vascular endothelial growth factor (VEGF) and angiogenic inhibitor pigment epithelium-derived factor (PEDF) were determined. Angiogenic activity of the aqueous humor was quantified by measuring its effect on the migration of capillary endothelial cells. In the aqueous fluid, VEGF levels were increased in diabetics (mean values: 501 vs. 367 pg/ml; p = 0.05), compared to controls. PEDF was found to be decreased in diabetics (mean values: 2080 vs. 5780 ng/ml; p = 0.04) compared to controls. In seven diabetic patients with proliferative retinopathy, the most profound finding was a significant decrease of the PEDF level (mean value: 237 ng/ml), whereas VEGF levels were comparable to diabetic patients without proliferation (mean value: 3153; p = 0.003). Angiogenic activity in samples of patients from the control group was generally inhibitory due to PEDF, and inhibition was blocked by neutralizing antibodies to PEDF. Likewise, in diabetics without proliferation, angiogenic activity was also blocked by antibodies to PEDF. We will demonstrate here that the level of the natural ocular anti-angiogenic agent PEDF is inversely associated with proliferative retinopathy. PEDF is an important negative regulator of angiogenic activity of aqueous humor. Our data may have implications for the development of novel regimens for diabetic retinopathy.

Low content of the natural ocular anti-angiogenic agent pigment epithelium-derived factor (PEDF) in aqueous humor predicts progression of diabetic retinopathy.

AIMS/HYPOTHESIS: Retinopathy is the most common microvascular complication of diabetes. Our aim was to address the predictive value of pro-angiogenic and anti-angiogenic markers for progression of retinopathy. METHODS: Aqueous humor was collected at cataract surgery from 32 diabetic patients who had no or very mild retinopathy (ETDRS stage 47B). This subgroup showed lower pigment epithelium-derived factor content when compared to non-progressors and control subjects. Migratory activity in samples of patients from the control group and in diabetic patients without progression was generally inhibitory due to pigment epithelium-derived factor. Inhibition was blocked by neutralizing antibodies to pigment epithelium-derived factor. In diabetic patients initial angiogenic activity was higher in those who later developed retinopathy (vs. controls p=0.00005; vs. no progressors p=0.0003). Both pigment epithelium-derived factor and migratory response predicted progression. CONCLUSION/INTERPRETATION: Pigment epithelium-derived factor is an important negative regulator of angiogenic activity of aqueous humor. Its content in the aqueous humor of diabetic patients strongly predicts who among them will develop progression of retinopathy.

Thrombospondin-1, vascular endothelial growth factor and fibroblast growth factor-2 are key functional regulators of angiogenesis in the prostate.

BACKGROUND: Prostate cells secrete many molecules capable of regulating angiogenesis; however, which of these actually function as essential regulators of neovascularization is not yet clear. METHODS: Functional angiogenic mediators secreted by normal and diseased prostate cells were identified using an in vitro angiogenesis assay. These factors were quantified by immunoblot or ELISA and localized in tissue by immunohistochemistry. RESULTS: Normal prostate epithelial cell secretions were anti-angiogenic due to inhibitory thrombospondin-1 (TSP-1) whereas this inhibitor was decreased in the pro-angiogenic secretions derived from benign prostatic hyperplasia (BPH) and cancer cells. This pro-angiogenic activity depended primarily on fibroblast growth factor-2 (FGF-2) and/or vascular endothelial growth factor (VEGF) whose secretion was increased. Immunolocalization studies confirmed that the changes detected in vitro also occurred in vivo. CONCLUSIONS: During disease progression in the prostate, production of TSP-1, the major inhibitor, is down-regulated while that of stimulatory FGF-2 and/or VEGF rise, leading to the induction of the new vessels necessary to support tumor growth.

Thrombospondin-1 gene expression affects survival and tumor spectrum of p53-deficient mice.

In vitro and in vivo data indicate that thrombospondin-1 (TSP1) inhibits tumor progression in several ways including direct effects on cellular growth and apoptosis in the stromal compartment. To evaluate the importance of TSP1 for the progression of naturally arising tumors in vivo, we have crossed TSP1-deficient mice with p53-deficient mice. In p53-null mice, the absence of TSP1 decreases survival from 160 +/- 52 days to 149 +/- 42 days. A log-rank test comparing survival curves for these two populations yields a two-sided P value of 0.0272. For mice that are heterozygous for the p53-null allele, survival is 500 +/- 103 days in the presence of TSP1 expression, and 426 +/- 125 days in its absence (P = 0.0058). Whereas TSP1 expression did not cause a measurable change in the incidence of the majority of tumor types, a statistically significant (P < or = 0.05) decrease in the incidence of osteosarcomas is observed in the absence of TSP1. To determine more directly if host TSP1 inhibits tumor growth, B16F10 melanoma and F9 testicular teratocarcinoma cells have been implanted in C57BL/6J and 129Sv TSP1-null mice, respectively. The B16F10 tumors grow approximately twice as fast in the TSP1-null background and exhibit an increase in vascular density, a decrease in the rate of tumor cell apoptosis, and an increase in the rate of tumor cell proliferation. Increased tumor growth is also observed in the absence of TSP1 on the 129Sv genetic background. These data indicate that endogenous host TSP1 functions as a modifier or landscaper gene to suppress tumor growth.

c-Jun N-terminal kinase activation is required for the inhibition of neovascularization by thrombospondin-1.

Thrombospondin-1 (TSP-1) is a potent inhibitor of angiogenesis that acts directly on endothelial cells via the CD36 surface receptor molecule to halt their migration, proliferation, and morphogenesis in vitro and to block neovascularization in vivo. Here we show that inhibitory signals elicited by TSP-1 did not alter the ability of inducers of angiogenesis to activate p42 and p44 mitogen-activated protein kinase (MAPK). Rather, TSP-1 induced a rapid and transient activation of c-Jun N-terminal kinases (JNK). JNK activation by TSP-1 required engagement of CD36, as it was blocked by antagonistic CD36 antibodies and stimulated by short anti-angiogenic peptides derived from TSP-1 that act exclusively via CD36. TSP-1 inhibition of corneal neovascularization induced by bFGF was severely impaired in mice null for JNK-1, pointing to a critical role for this stress-activated kinase in the inhibition of neovascularization by TSP-1.

In vivo mechanisms by which tumors producing thrombospondin 1 bypass its inhibitory effects.

Thrombospondin 1 (TSP1) is a multifunctional protein able to activate TGFbeta and to inhibit angiogenesis in vivo. Although usually thought of as an inhibitor of tumor growth, TSP1 may sometimes be present at high levels during tumor progression, suggesting that tumors can eventually overcome their anti-tumor effects. Using a tet-repressible expression system, we demonstrate that murine TSP1 delayed the onset of tumor growth when produced in the tumor bed by rat fibrosarcoma tumor cells or by stromal fibroblasts coinjected with unmodified C6 glioma tumor cells. Yet upon prolonged exposure to TSP1, tumors came to grow at the same rate in the presence as in the absence of TSP1 and transplantation experiments showed that they had become insensitive to inhibition by TSP1 in both syngeneic and immune compromised hosts. Tumor resistance to TSP1 developed as a result of the in vivo outgrowth of pre-existing tumor cell variants that (1) secreted increased amounts of angiogenic factors that counterbalanced the inhibitory effect of TSP1 on neovascularization and (2) grew more efficiently in the presence of TSP1-activated TGFbeta. These results indicate that prolonged and continuous local delivery of a single multifunctional angiogenesis inhibitor like TSP1 to fast-growing tumors can lead to tumor resistance in vivo by fostering the outgrowth of subpopulations that are a by-product of the genetic instability of the tumor cells themselves.

The role of hypoxia and p53 in the regulation of angiogenesis in bladder cancer.

PURPOSE: Our previous studies defined thrombospondin-1 (TSP-1) and vascular endothelial growth factor (VEGF) as the primary mediators of angiogenesis in the bladder and the loss of inhibitory TSP-1 as a key event in the transition to an angiogenic phenotype during bladder cancer development. We evaluated the role of p53, which is commonly inactivated in bladder cancer, and hypoxia in the regulation of angiogenesis in the bladder. MATERIALS AND METHODS: The p53 status was modulated in normal urothelial and bladder cancer cells, and conditioned media was collected under normal oxygen or hypoxic (0.5% O2) conditions. Angiogenic activity was evaluated with the endothelial cell migration assay, and the levels of secreted TSP-1 and VEGF were determined by Western blot analysis and enzyme-linked immunosorbent assay, respectively. RESULTS: Retroviral mediated expression of the E6 oncoprotein reduced wild-type p53 levels in normal urothelial cells by greater than 90% but did not significantly alter TSP-1 or VEGF levels, while total inductive and inhibitory activities remained unchanged. Adenoviral mediated expression of wild-type p53 was confirmed in 4 bladder cancer cell lines by Western blot analysis for p53 and its downstream effector protein p21 (2.5 to 5.0-fold increase). TSP-1 levels remained unchanged but the levels of secreted VEGF in the high grade UMUC-3 and 253J cell lines were significantly decreased 5 to 50-fold and a corresponding decrease in net angiogenic activity was observed. However, (increased expression) of p53 had no effect on the angiogenic activity of the low grade RT4 or high grade HT1376 bladder cancer cells. Hypoxia converted normal urothelial cell derived conditioned media from anti-angiogenic to angiogenic and increased the angiogenic activity of bladder cancer cell derived conditioned media. This change was due to 2.5 to 6-fold hypoxic up-regulation of VEGF because the expression of inhibitory TSP-1 was not significantly altered. CONCLUSIONS: Our results suggest that p53 does not regulate angiogenesis in the bladder in the setting of an otherwise normal genome and gene therapy with wild-type p53, which is currently being studied for this cancer, may have only limited effects on angiogenesis. In contrast, hypoxia regulates angiogenesis in this system, primarily through its effects on VEGF.

Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor.

Aberrant blood vessel growth in the retina that underlies the pathology of proliferative diabetic retinopathy and retinopathy of prematurity is the result of the ischemia-driven disruption of the normally antiangiogenic environment of the retina. In this study, we show that a potent inhibitor of angiogenesis found naturally in the normal eye, pigment epithelium-derived growth factor (PEDF), inhibits such aberrant blood vessel growth in a murine model of ischemia-induced retinopathy. Inhibition was proportional to dose and systemic delivery of recombinant protein at daily doses as low as 2.2 mg/kg could prevent aberrant endothelial cells from crossing the inner limiting membrane. PEDF appeared to inhibit angiogenesis by causing apoptosis of activated endothelial cells, because it induced apoptosis in cultured endothelial cells and an 8-fold increase in apoptotic endothelial cells could be detected in situ when the ischemic retinas of PEDF-treated animals were compared with vehicle-treated controls. The ability of low doses of PEDF to curtail aberrant growth of ocular endothelial cells without overt harm to retinal morphology suggests that this natural protein may be beneficial in the treatment of a variety of retinal vasculopathies.

Pigment epithelium-derived factor (PEDF) in neuroblastoma: a multifunctional mediator of Schwann cell antitumor activity.

Neuroblastoma is notable for its cellular heterogeneity and unpredictable outcome. Tumors are a variable mixture of primitive malignant neuroblasts, more differentiated ganglionic cells, Schwann and endothelial cells. Although often fatal, neuroblastomas can spontaneously regress, possibly due to favorable autocrine and paracrine interactions among these cells. Here, pigment epithelium-derived factor (PEDF), a potent inhibitor of angiogenesis and inducer of neural differentiation, is shown to be produced by ganglionic cells and Schwann cells, but not by more primitive tumor cells. Although undifferentiated neuroblastoma tumor cell secretions were angiogenic primarily due to vascular endothelial growth factor, secretions of Schwann cells were anti-angiogenic due to PEDF. In addition, PEDF was the major factor responsible for Schwann cell's ability to induce tumor cell differentiation in vitro and recombinant PEDF had the same effect in vitro and in vivo. Both the growth and the survival of Schwann cells were enhanced by PEDF. Thus PEDF may serve as a multifunctional antitumor agent in neuroblastomas, inhibiting angiogenesis while promoting the numbers of Schwann cells and differentiated tumor cells that in turn produce PEDF, suggesting that its clinical administration could stimulate a multifaceted antitumor feedback loop with the potential to limit and possibly regress tumor growth.

Smad4/DPC4-mediated tumor suppression through suppression of angiogenesis.

Smad4/DPC4 (deleted in pancreatic carcinoma, locus 4) is a tumor suppressor gene lost at high frequency in cancers of the pancreas and other gastrointestinal organs. Smad4 encodes a key intracellular messenger in the transforming growth factor beta (TGF-beta) signaling cascade. TGF-beta is a potent inhibitor of the growth of epithelial cells; thus, it has been assumed that loss of Smad4 during tumor progression relieves this inhibition. Herein, we show that restoration of Smad4 to human pancreatic carcinoma cells suppressed tumor formation in vivo, yet it did not restore sensitivity to TGF-beta. Rather, Smad4 restoration influenced angiogenesis, decreasing expression of vascular endothelial growth factor and increasing expression of thrombospondin-1. In contrast to the parental cell line and to control transfectants that produced rapidly growing tumors in vivo, Smad4 revertants induced small nonprogressive tumors with reduced vascular density. These data define the control of an angiogenic switch as an alternative, previously unknown mechanism of tumor suppression for Smad4 and identify the angiogenic mediators vascular endothelial growth factor and thrombospondin-1 as key target genes.

A pilot trial of suramin in metastatic breast cancer to assess antiangiogenic activity in individual patients.

Suramin is a polysulfonated naphthylurea with multiple potential mechanisms of action against tumors, including the ability to bind growth factors known to promote tumor angiogenesis. Using an established fixed dosing scheme for the administration of suramin in patients, a pilot study was conducted in patients with progressive, metastatic breast cancer. The primary objective of this trial is to define the effect of suramin on the angiogenic activity in individual patients using in vitro laboratory assays. The secondary objective was to assess the antitumor effect of suramin in a population of metastatic breast cancer patients. No objective tumor responses were observed in any of the 9 patients who received treatment with suramin, however 1 patient did maintain stable disease status. The strength of angiogenic activity present in patient samples was assessed by testing patient plasma in the capillary endothelial cell migration assay. Angiogenic activity followed over time was lowest in patients with the highest suramin concentrations and highest in patients with the lowest suramin concentrations. We conclude that it is feasible to continually monitor the activity of antiangiogenic agents in individual patients without relying on clinical tumor response.

Maspin is an angiogenesis inhibitor.

Maspin, a unique member of the serpin family, is a secreted protein encoded by a class II tumor suppressor gene whose downregulation is associated with the development of breast and prostate cancers. Overexpression of maspin in breast tumor cells limits their growth and metastases in vivo. In this report we demonstrate that maspin is an effective inhibitor of angiogenesis. In vitro, it acted directly on cultured endothelial cells to stop their migration towards basic fibroblast growth factor and vascular endothelial growth factor and to limit mitogenesis and tube formation. In vivo, it blocked neovascularization in the rat cornea pocket model. Maspin derivatives mutated in the serpin reactive site lost their ability to inhibit the migration of fibroblasts, keratinocytes, and breast cancer cells but were still able to block angiogenesis in vitro and in vivo. When maspin was delivered locally to human prostate tumor cells in a xenograft mouse model, it blocked tumor growth and dramatically reduced the density of tumor-associated microvessels. These data suggest that the tumor suppressor activity of maspin may depend in large part on its ability to inhibit angiogenesis and raise the possibility that maspin and similar serpins may be excellent leads for the development of drugs that modulate angiogenesis.

Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1.

Thrombospondin-1 (TSP-1) is a naturally occurring inhibitor of angiogenesis that limits vessel density in normal tissues and curtails tumor growth. Here, we show that the inhibition of angiogenesis in vitro and in vivo and the induction of apoptosis by thrombospondin-1 all required the sequential activation of CD36, p59fyn, caspase-3 like proteases and p38 mitogen-activated protein kinases. We also detected increased endothelial cell apoptosis in situ at the margins of tumors in mice treated with thrombospondin-1. These results indicate that thrombospondin-1, and possibly other broad-spectrum natural inhibitors of angiogenesis, act in vivo by inducing receptor-mediated apoptosis in activated microvascular endothelial cells.

Pigment epithelium-derived factor: a potent inhibitor of angiogenesis.

In the absence of disease, the vasculature of the mammalian eye is quiescent, in part because of the action of angiogenic inhibitors that prevent vessels from invading the cornea and vitreous. Here, an inhibitor responsible for the avascularity of these ocular compartments is identified as pigment epithelium-derived factor (PEDF), a protein previously shown to have neurotrophic activity. The amount of inhibitory PEDF produced by retinal cells was positively correlated with oxygen concentrations, suggesting that its loss plays a permissive role in ischemia-driven retinal neovascularization. These results suggest that PEDF may be of therapeutic use, especially in retinopathies where pathological neovascularization compromises vision and leads to blindness.

Keratinocyte growth factor induces angiogenesis and protects endothelial barrier function.

Keratinocyte growth factor (KGF), also called fibroblast growth factor-7, is widely known as a paracrine growth and differentiation factor that is produced by mesenchymal cells and has been thought to act specifically on epithelial cells. Here it is shown to affect a new cell type, the microvascular endothelial cell. At subnanomolar concentrations KGF induced in vivo neovascularization in the rat cornea. In vitro it was not effective against endothelial cells cultured from large vessels, but did act directly on those cultured from small vessels, inducing chemotaxis with an ED50 of 0.02-0.05 ng/ml, stimulating proliferation and activating mitogen activated protein kinase (MAPK). KGF also helped to maintain the barrier function of monolayers of capillary but not aortic endothelial cells, protecting against hydrogen peroxide and vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induced increases in permeability with an ED50 of 0.2-0.5 ng/ml. These newfound abilities of KGF to induce angiogenesis and to stabilize endothelial barriers suggest that it functions in microvascular tissue as it does in epithelial tissues to protect them against mild insults and to speed their repair after major damage.

Three distinct D-amino acid substitutions confer potent antiangiogenic activity on an inactive peptide derived from a thrombospondin-1 type 1 repeat.

Mal II, a 19-residue peptide derived from the second type 1 properdin-like repeat of the antiangiogenic protein thrombospondin-1 (TSP-1), was inactive in angiogenesis assays. Yet the substitution of any one of three L-amino acids by their D-enantiomers conferred on this peptide a potent antiangiogenic activity approaching that of the intact 450-kDa TSP-1. Substituted peptides inhibited the migration of capillary endothelial cells with an ED50 of 8.5 nM for the D-Ile-15 substitution, 10 nM for the D-Ser-4 substitution, and 0.75 nM for the D-Ser-5 substitution. A peptide with D-Ile at position 15 could be shortened to its last seven amino acids with little loss in activity. Like whole TSP-1, the Mal II D-Ile derivative inhibited a broad range of angiogenic inducers, was selective for endothelial cells, and required CD36 receptor binding for activity. A variety of end modifications further improved peptide potency. An ethylamide-capped heptapeptide was also active systemically in that when injected i.p. it rendered mice unable to mount a corneal angiogenic response, suggesting the potential usefulness of such peptides as antiangiogenic therapeutics.

The anticancer drug edelfosine is a potent inhibitor of neovascularization in vivo.

Edelfosine is an alkyl-lysophospholipid that acts as an anticancer agent in vivo. To test the hypothesis that part of its antineoplastic activity may be due to its ability to inhibit the neovascularization on which the progressive growth of all tumors depends, we evaluated edelfosine in vitro and in vivo for antiangiogenic activity. Edelfosine acted directly on cultured capillary endothelial cells, inhibiting their migration toward the angiogenic factor, basic fibroblastic growth factor (bFGF), at doses of 8-200 nM. When given systemically to rats (20 mg/kg i.p. twice daily), edelfosine was well tolerated and antiangiogenic. The majority of treated animals became unable to mount a corneal neovascular response to a pellet releasing bFGF, whereas vigorous vessel ingrowth was seen in untreated controls.

Multiple forms of angiostatin induce apoptosis in endothelial cells.

Angiostatin is a circulating inhibitor of angiogenesis generated by proteolytic cleavage of plasminogen. In this study we have used recombinant human and murine angiostatins (kringles 1-4) as well as native human angiostatin (prepared by elastase digestion of plasminogen [kringles 1-3] or by plasmin autocatalysis in the presence of a free sulfhydryl donor [kringles 1-4]). We report that angiostatin reduces endothelial cell number in a 4-day proliferation assay without affecting cell cycle progression into S-phase (as determined by bromodeoxyuridine labeling). This suggested that the reduction in cell number in the proliferation assay might in part be due to cytotoxicity. This was confirmed by the observation that ethidium homodimer incorporation (a measure of plasma membrane integrity) into endothelial cells was increased by angiostatin in a manner similar to that seen with tumor necrosis factor- (TNF-) and transforming growth factor-beta1 (TGF-beta1), both of which induce apoptosis in endothelial cells. In contrast to TNF- and TGF-beta1, angiostatin did not induce cytotoxicity in human MRC-5 fibroblast, rat smooth muscle, canine MDCK epithelial, or murine B16-F10 melanoma cell lines. Angiostatin-induced apoptosis was confirmed by endothelial cell nuclear acridine orange incorporation as well as by annexin V and TUNEL staining. These in vitro findings point to endothelial cell apoptosis as a mechanism for the antiangiogenic effect of angiostatin in vivo.

Retinoic acid and interferon alpha act synergistically as antiangiogenic and antitumor agents against human head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy in which multiple independent lesions develop over time throughout the mucosa of the upper aerodigestive tract. Therefore, the comprehensive treatment of this neoplasm must include a chemopreventive arm to hold premalignant lesions in check, a role well-suited to antiangiogenic agents. Retinoic acid (RA) and interferon alpha (IFN-alpha), drugs with known biological activity against HNSCC when used individually, are also inhibitors of angiogenesis. Here we show that they are remarkably synergistic antiangiogenic agents able to inhibit both the growth and the neovascularization of HNSCC injected into the floor of the mouth of nude mice. The mechanism of action of these drugs as antiangiogenic agents was 2-fold. They decreased the angiogenic activity of the tumor cells, and they caused the endothelial cells to become refractory to inducers of angiogenesis. When tumor cells were treated in vitro with IFN-alpha A/D, there was a dramatic drop in their secretion of interleukin-8, the major angiogenic factor produced by these tumors. When combined with RA, which causes tumor cells to secrete an inhibitor of angiogenesis, there was a synergistic inhibition of both tumor cell growth and secreted angiogenic activity. The combination of RA and IFN-alpha also acted synergistically on endothelial cells by reducing their responsiveness to both interleukin-8 and tumor conditioned media. Doses of each drug could be reduced by two logs without loss of activity. When animals bearing human HNSCC tumor cells were treated systemically with a combination of RA and IFN-alpha A/D at doses that were ineffective when used alone, dramatic decreases in both tumor growth and tumor angiogenesis were seen. These data suggest that the use of antiangiogenic mixtures may be a particularly effective way to design future chemoprevention protocols against HNSCC.

Inhibition of angiogenesis by interleukin 4.

Interleukin (IL)-4, a crucial modulator of the immune system and an active antitumor agent, is also a potent inhibitor of angiogenesis. When incorporated at concentrations of 10 ng/ml or more into pellets implanted into the rat cornea or when delivered systemically to the mouse by intraperitoneal injection, IL-4 blocked the induction of corneal neovascularization by basic fibroblast growth factor. IL-4 as well as IL-13 inhibited the migration of cultured bovine or human microvascular cells, showing unusual dose-response curves that were sharply stimulatory at a concentration of 0.01 ng/ml but inhibitory over a wide range of higher concentrations. Recombinant cytokine from mouse and from human worked equally well in vitro on bovine and human endothelial cells and in vivo in the rat, showing no species specificity. IL-4 was secreted at inhibitory levels by activated murine T helper (TH0) cells and by a line of carcinoma cells whose tumorigenicity is known to be inhibited by IL-4. Its ability to cause media conditioned by these cells to be antiangiogenic suggested that the antiangiogenic activity of IL-4 may play a role in normal physiology and contribute significantly to its demonstrated antitumor activity.